ABSTRACT Antibodies possess exquisite specificity and affinity for their antigens and as a consequence, Positron Emission Tomography (PET) using targeted antibodies is a molecular imaging technique at the forefront of cancer diagnosis and treatment management. Zirconium-89 (89Zr), a positron-emitting radionuclide, possesses excellent physical properties for PET imaging when paired with antibodies. The 78.41 hr half-life of 89Zr is a near perfect match to the localization time of long circulating IgG antibodies. There has been increased interest and a dramatic rise in the use of 89Zr as a PET radiometal paired with antibodies over the past ten years. These ongoing clinical studies and all pre-clinical studies use the current standard bifunctional ligand (also called a bifunctional chelator) for 89Zr: Desferrioxamine B (DFO). Although image quality is generally very good, DFO is not the optimal ligand for 89Zr. This is revealed by some subtle bone and non-target uptake of the radioisotope due to release of osteophilic 89Zr from DFO. There is therefore a need to develop an improved bifunctional ligand for 89Zr that will significantly improve 89Zr-antibody PET imaging by providing an improved alternative to DFO, reducing absorbed doses to healthy tissues and therefore safer PET imaging and enhanced image quality. This R21 project is designed with the overall goal to form superior bifunctional ligands for conjugation to antibodies and for stable chelation of 89Zr. Such a bifunctional ligand will eliminate 89Zr loss from the chelate in vivo and uptake in bone and non-target issue. In essence, this new bifunctional ligand will facilitate safer and improved PET imaging with 89Zr-labeled antibodies. Under the auspices of this R21, we will synthesize and characterize a series of bifunctional ligands with varying linker chemistries for the chelation of 89Zr and conjugation to antibodies. The synthetic effort will be paired with in silico DFT calculations and molecular dynamics simulations to identify the coordination environment, comparative stabilities and inform on the ease of radiometallation. We will evaluate the ligands synthesized when conjugated to trastuzumab (as a well-studied model system) and radiolabeled with 89Zr to determine their pharmacokinetics and stabilities. The 89Zr-ligand-trastuzumab constructs will be evaluated to determine their stability, biodistribution, and overall utility as PET imaging agents compared to 89Zr-DFO-trastuzumab